chlorin-p6 and Necrosis

Our previous studies in hamster cheek pouch model have shown that chlorin p (6) (Cp (6)), a chlorophyll derivative is a suitable photosensitizer for photodynamic treatment (PDT) of small tumors (<5 mm). However, for bigger tumors, the accumulation of Cp (6) was inadequate, which compromised the effectiveness of PDT. The purpose of present study was to investigate the possibility of improving the cellular uptake of Cp (6) by conjugating it to histamine, a biogenic amine that is known to modulate tumor growth and development via cell surface receptors.. The conjugate of Cp (6) and histamine (Cp (6)-his) was prepared by carbodiimide coupling reaction. Cellular uptake, intracellular localization and cytotoxicity of both Cp (6) and its conjugate were investigated in two human oral cancer cell lines (4451 and NT8e). The percentage of necrotic and apoptotic cells after PDT were also estimated using Hoechst 33342-propidium iodide staining.. In both the cell line, the cellular uptake of Cp (6)-his was found to be ~10 times higher when compared to Cp (6). Histamine led to a slight increase in intracellular uptake of Cp (6)-his, whereas ranitidine, a histamine H2 receptor antagonist, and incubation at lower temperature (~15°C) led to its inhibition, suggesting that uptake of Cp (6)-his is receptor mediated. Results on western blot confirmed the presence of H2 receptor in both the cell line. Observations on intracellular localization revealed that unlike Cp (6), which localized on multiple sites, Cp (6)-his showed localization on the cell membrane and around the perinuclear region. Moreover, the phototoxicity induced by Cp (6)-his was ~4 times higher when compared to Cp (6) in both the cell lines. There was, however, no significant difference in the mode of cell death.. Results suggest that conjugating Cp (6) with histamine can help improve the effectiveness of PDT in oral cancer cells by enhancing its intracellular delivery.

Topics: Apoptosis; Biological Transport; Carbodiimides; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cold Temperature; Cross-Linking Reagents; Histamine; Histamine H2 Antagonists; Humans; Kinetics; Mouth Neoplasms; Necrosis; Osmolar Concentration; Photochemotherapy; Photosensitizing Agents; Porphyrins; Ranitidine; Receptors, Histamine H2

A new photosensitizer, LCP, a combination of lysyl chlorin p6 and chlorin p6, was synthesized and tested for effectiveness in photodynamic therapy using s.c. implanted 9L glioma tumors in rats. Tumors were irradiated with 664-nm light 4 h after LCP injection. Mean intratumoral temperature elevations were less than 4 degrees C using a power density of 50 mW/cm2 for 33.3 min (100 J/cm2). Subsequent experiments examining histological changes and tumor regrowth used a power density of 50 mW/cm2 and total energy densities of 25, 50, and 100 J/cm2. Microscopically, an energy density-dependent coagulation necrosis of tumor cells occurred in treated tumors. Long term inhibition of tumor growth was achieved only at an energy density of 100 J/cm2. Side effects of treatment were seen only in the irradiated area and consisted of coagulation necrosis of normal tissues in rats treated at 50 and 100 J/cm2, including severe skin necrosis. Exposure of rats to fluorescent room light did not cause any macroscopically detectable skin damage. Our data indicate that photodynamic destruction of s.c. 9L glioma tumors using LCP as a photosensitizer results in significant tumor growth inhibition and that further study of LCP is warranted.

Topics: Animals; Drug Screening Assays, Antitumor; Glioma; Lysine; Male; Necrosis; Neoplasm Transplantation; Photochemotherapy; Porphyrins; Rats; Rats, Inbred F344; Skin; Skin Neoplasms